Project Summary This ?High risk-High reward? proposal will test a novel and unique Tobacco Mosaic Virus (TMV) based vaccine delivery platform for development of subunit vaccines. We propose to use tularemia as a disease model to demonstrate the proof of efficacy of TMV-based subunit vaccines. This novel approach has several advantages over the current approaches employed for development of an effective vaccine against tularemia. This approach is safe, scalable, affordable, potent and capable of inducing strong innate and adaptive immune responses. Tularemia is a fatal human disease caused by highly virulent and category A select agent Francisella tularensis (Ft). No FDA approved vaccine is currently available for prophylaxis of tularemia. The history of Ft weaponization and the recent concerns about its use as a bioweapon/bioterror agent has potentiated an urgent need for the development of a safe and effective vaccine. Nonspecific symptoms of tularemia and the engineered antibiotic resistant strains undermine therapeutic options available for the treatment of tularemia. In the last 100 years since the discovery of F. tularensis, three broad approaches comprising of killed whole cell, live attenuated and subunit vaccines have been employed for vaccine development, but none of these have been successful. The challenges thus far in development of multivalent subunit vaccines have been the availability of suitable approaches for consistent preparation and efficient delivery of multiple antigens through mucosal routes. As a proof-of-concept of the efficacy of TMV-conjugated tularemia vaccine, our published and preliminary studies demonstrate that multiple protective antigens of Ft conjugated to TMV can be administered intranasally in mice without any adverse reactions, induce a potent adaptive immune response to Ft antigens and most importantly provide 100% protection in mice against intranasal challenge with Ft LVS. The goals of this proposal are to establish the efficacy of TMV- conjugates as an efficient and effective platform for simultaneous delivery of multiple protective antigens of Ft and to develop an effective vaccine for prevention of tularemia caused by highly virulent and Tier 1 Category A Select Agent Ft SchuS4 strain. Two specific aims are proposed to achieve these goals. Aim1 will be involved in production and testing the protective efficacy of Ft TMV-conjugate vaccine against Ft SchuS4 challenge and Aim 2 will investigate the mechanism and correlates of immune protection in mice vaccinated with fully protective Ft TMV- conjugate vaccine. At the conclusion of these studies, we intend to identify a lead tularemia vaccine formulation that could be developed into a safe, affordable and potent human vaccine. The results from the proposed studies will also establish the utility of TMV as novel and unique a platform for simultaneous delivery of multiple protective antigens that could be adopted for development of multivalent subunit vaccines against other infectious diseases.